Estradiol, which acts through the Estrogen Receptor alpha (ER), is essential for the development and homeostasis of female reproductive function, exerting critical control of many transcriptional programs, including these regulatory proliferatio, and metabolism. The molecular mechanism used by ER to mediate coactivator/corepressor exchanges in gene activation has been reasonably well elucidated. ER, chromatin modifiers and dynamic positioning of particular loci are all essential for establishing precise transcriptionl regulation and achieving the correct patterns of gene expression. However, it is not clear how these factors are spatially regulated in distinct subnuclear structures and how this regulation may contribute to gene regulation. ) Our recent findings demonstrated a signal-induced relocation of the transcription units from a transcriptional repressive compartment to a permissive environment. I hypothesize that non-coding RNAs (ncRNAs) TUG1 and NEAT2 control ER target genes to relocate from the transcriptional repressive Polycomb bodies (PcGs) to the gene activation milieu of the interchromatin granules, by selectively interacting with methylated and unmethylated Polycomb 2 protein (Pc2) present on ER target gene promoters. To address this long-term goal, I propose to investigate the hypothesis from three specific aims: 1) to define non- histone methylation/demethylation events and ncRNAs as a novel molecular strategy responsible for genome- wide ER transcriptional programs; 2) to determine the potential role of two ncRNAs, TUG1 and NEAT2, located in PcGs and interchromatin granules, respectively, in controlling relocation of ER target genes between these two subnuclear structures, depending on the status of Pc2 methylation in response to estrogen; 3) to investigate the potential allosteric role of ncRNA in modulating the ability of readers of the histone code, such as Pc2, to recognize histone tail modifications. To achieve these aims, I propose to define the genome-wide location of methylated vs. unmethylated Pc2 by ChIP-Seq analysis using specific antibodies and the functional roles of Pc2 or other active readers and ncRNAs in controlling the ER transcriptome by a combination of GRO-Seq, RNA-Seq analysis and siRNA knockdown strategies. I will also investigate the potential relocalization of ER-regulated genes from PcG bodies to interchromatin granules upon E2 stimulation and the underlying mechanisms, by which the binding of TUG1 or NEAT2 ncRNAs directs the preference of Pc2 recognition of histone tail modification. Taken together, the proposed study evaluates a new concept that ER target gene loci relocate between functionally distinct nuclear architectural structures and the roles of several novel modulators that play a critical role in ER target gene regulation. These findings will provide innovative targets for drugs development for a number of intriguing therapeutic implications. Based on the extensive preliminary data, I am confident of accomplishing these aims in the period of the award and emerging as an Independent Investigator. PUBLIC HEALTH RELEVANCE: It has been well established that estrogen plays important roles in promoting mammary gland development, regulating transcription and metabolism, and many pathological conditions. While much is understood about actions of Estrogen Receptor, additional unexplored molecular strategies appear to contribute to the critical aspect of estrogen function. The proposed studies in this application dissect the control of estrogen receptor target gene expression from an innovative view that incorporates three-dimensional context of nuclear architecture, epigenetic modifications and novel non-coding RNAs, which will uncover new approaches to treat estrogen-related disease using small RNAs to target ncRNA transcripts.